Recently, evidence was presented that the Cln2-Cdc28 cyclin-dependent kinase inhibits the mating factor signal transduction pathway by interfering with the function of Ste20, and that this inhibition correlates with Cln2-dependent in vivo phosphorylation of Ste20. We used MALDI-TOF-MS and LC-ESI-ion trap-MS/MS analysis of unlabeled full length Ste20 as well as a truncated form spanning residues 496-939 (Ste20trunc) to identify 13 sites that were phosphorylated in vivo. Here, we apply the our newly developed method for quantitating changes in the level of phosphorylation to specifically identify Cln2-dependent in vivo phosphorylation sites in Ste20. We use the data to test the hypothesis that Cln2-dependent phosphorylation of Ste20 brings about inhibition of the mating factor transduction pathway.